Defects in the degradation of toxic proteins and aberrant accumulation thereof as well as transynaptic exosomal spreading of toxic species and dysregulation of cholesterol levels have been increasingly linked to the pathogenesis of neurodegenerative diseases. Interestingly, all these pathways are regulated by the late endosomes/lysosomes (LE/Lys)-resident bis(monoacylglycerol)phosphate (BMP). There, this fusogenic lipid controls the budding of intra-lumenal vesicles (ILVs) and thus regulates the correct addressing of cargoes to either intracellular degradation in LE/Lys or to extracellular export in exosomes. It also controls cholesterol levels, through the stimulation of its egress from LE/Lys. Indeed, BMP has been shown to be upregulated in a number of neurodegenerative disorders, such as Alzheimer's disease and dementia with Lewy bodies. The ability to modulate BMP levels would be of crucial interest not only in understanding the pathogenesis of these disorders but also as a potential therapeutic tool. The key obstacle to this approach is the fact that the key enzymes regulating BMP synthesis remain unknown. The first and limiting step of the BMP biosynthetic pathway is the conversion of phosphatidylglycerol (PG) into lyso-PG by a phospholipase A2 (PLA2). Our preliminary work focused on lysosomal PLA2 (LPLA2) because of its putative localization. We found in a liposomes assay that LPLA2 was sufficient to catalyze the conversion of PG into lyso-PG. We also observed that knocking-down LPLA2 in HeLa cells resulted in a decrease of the levels of BMP. Intriguingly, our immunostaing experiments revealed that LPLA2 did not overlap with LAMP1-positive LE/Lys but rather was an endoplasmic reticulum (ER)-resident protein that is enriched at ER-LE/Lys membrane contact sites (MCS). The LE-resident ORP1L was reported to regulate ER-LE MCS that are enriched with VAMP-associated protein A (VAPA) on the ER side, through its sensing of cholesterol levels and we found that VAPA and LPLA2 partially colocalized and contacted RAB7-positive LE. The main goals of this proposal are thus to test whether (i) LPLA2 plays an unexpected role in the control of BMP synthesis in HeLa cells and cultured primary neurons and (ii) the actual intracellular localization of LPLA2 at ER-LE/Lys MCS is controlled through the sensing of cholesterol levels in the limiting membrane of LE/Lys by ORP1L. Finally, we focused the current proposal on Alzheimer's disease (AD) as both cholesterol and BMP are increased in the cortices of AD patients. Because BMP regulates cholesterol levels and based on our previous work suggesting that cholesterol accumulation could cause not only amyloid pathology but also Tau pathology, we postulate that (iii) finding new regulators of BMP synthesis in a cellular model of AD, i.e. human embryonic stem cell (hESC)-derived neurons, could be an attractive way to delineate novel drug targets for Alzheimer's disease. The results of this proposal will unravel the mechanisms underlying the regulation of BMP synthesis in normal and diseased states and pave the way for the generation of exploratory and/or therapeutic tools,